ABSTRACT Neuronal early endosome pathology is among the earliest disease-specific features of Alzheimer?s disease (AD) and has been mechanistically linked to elevated levels of -site cleaved carboxyl-terminal fragment ( CTF) of the amyloid precursor protein (APP). Pathological features of the AD early endosome phenotype include rab5 hyperactivation, downstream disruption of the endosomal-lysosomal (EL) pathway, and a failure of endosome-mediated trophic support. Our preliminary findings show that expression of the ApoE4 allele, the greatest genetic-risk factor for AD, leads to alterations in neuronal early endosomes in ApoE4 mice (Core B). Given that alterations in cholesterol metabolism in the brain have been suggested to play a role in AD and that we find increased CTF levels in the brain with ApoE4 expression, we will test the hypothesis that ApoE4 drives CTF and cholesterol alterations that interact to promote endosomal dysfunction and downstream lysosomal network (LN) pathology ((Project 2 (P2), P3 and P4). We will examine the lipidome and proteome of brain endosomal fractions, isolated from ApoE4 expressing mice and humans (Core C) in order to identify the molecular mechanism driving ApoE4 EL pathway alterations and neuronal vulnerability. We will use direct manipulation of cholesterol in vitro and dietary challenge in vivo and manipulation of CTF levels by BACE1 downregulation to test the hypothesis that synergy between these molecules contributes to the pathological ApoE4-driven LN phenotype. At-risk neuronal populations will be identified and examined using gene expression analysis (P4) to elucidate the molecular mechanism of LN vulnerability. Behavioral assays will be used to demonstrate cognitive consequences (Core C). Given the importance of endosome-to-Golgi recycling as an exit pathway from early endosome and the recent recognition that retromer dysfunction may contribute to AD pathobiology, we have undertaken preliminary studies to determine whether manipulation of the CTF can rescue early endosomal pathology by stimulating the retromer pathway. We found that binding antibodies specifically to the CTF in living cells can increase retromer-containing vesicles while reducing early endosomal pathology. We will test the novel hypothesis that promoting CTF trafficking though the endosome- to-Golgi recycling pathway reduces pathogenic CTF endosome-signaling and rescues pathological changes in early endosomes and the neurodegenerative cascade this initiates. This proposal examines multiple trafficking routes from the early endosome as well as the consequences of early endosomal dysfunction, including impaired endosome-mediated trophic support, and, in collaboration with all of the other Projects, downstream pathological LN changes. This represents a conceptually innovative approach to AD risk and pathology while exploring novel molecular mechanisms ? including CTF trafficking and signaling, the role of cholesterol in CTF function, and interacting trafficking signals and cascades at the early endosome and retromer ? that drive this pathology.